Our long-term goal is to develop potent agents for prevention and treatment of anthrax and methicillinresistant S. aureus (MRSA) by engineering phage endolysins. PlyG, a lysin of 25 kDa molecular weight encoded by gamma phage. PlyG contains a T7 lysozyme-like catalytic domain capable of hydrolyzing B. anthracis cell wall peptidoglycan, resulting in bacterial cell lysis, attached to a -75 amino acid C- terminal domain. The C-terminal domain is a dimeric carbohydrate recognition module that targets the enzyme specifically to vegetative B. anthracis cells and germinating spores. We hypothesize that full- length PlyG exists in a monomeric inactive state stabilized by specific contacts between the N- and C-terminal domains, and that binding of the C-terminal (regulatory) domain to carbohydrates unique to the B. anthracis cell wall releases the autoinhibitory interaction and promotes formation of the fully active, dimeric PlyG enzyme. Phage endolytic enzymes like PlyG have the potential to serve as novel and powerful antibiotic agents, termed 'enzybiotics'. We broadened this approach to include MRSA by assembling an enzybiotic from other phage lysins with specific S. aureus activity. ClyS is a chimeric protein containing an N-terminal catalytic domain and a C-terminal cell wall targeting domain. The N-terminal catalytic domain is an endopeptidase of 184 amino acids and the C-terminal cell wall targeting domain is 94 residues. In aim 1, we will determine the NMR structure of full-length (inactive) PlyG to reveal the molecular basis of lysin autoinhibition for this class of antibacterial enzymes, and solve structures of the ClyS lysin and its component domains. In Aim 2, the basis for specific anthrax and MRSA targeting will be elucidated using NMR to monitor interactions between cell wall components and the PlyG and ClyS binding domains. Aim 3 will exploit this structural knowledge to engineer isoforms of PlyG and ClyS with enhanced stability in vivo. Because the bacterium must alter the basic construction of the cell wall to evade an enzybiotic, the probability that PlyG- and ClyS- resistant strains will emerge is low. These studies will provide important mechanistic insights into a novel class of antibacterial compounds moving toward clinical application.